IS10-element alters GFP-ATRX localization and incorporation into chromatin. (A) HEK-293T cells were transiently transfected with IS10-GFP-ATRX (left) or IF-GFP-ATRX (right). The localization of GFP-ATRX was determined by confocal fluorescence microscopy 72 hours post-transfection. Representative images are shown. Dashed lines on the highlighted cells indicate nuclei area. IS10-GFP-ATRX displays a diffuse nuclear and cytoplasmic localization. Conversely, ATRX-GFP has a nuclear localization in the IF-GFP-ATRX transfected cells with a focal pattern. (B) Western analysis of whole cell protein extracts from the IS10-GFP-ATRX and IF-GFP-ATRX transfections against GFP (top) and actin (bottom). Bands corresponding to IF-GFP-ATRX and IS10-GFP-ATRX products are indicated with arrows. (C) Chromatin extracts from the IS10-GFP-ATRX and IF-GFP-ATRX transfections were analyzed with GFP antibodies (top). Bands corresponding to IF-GFP-ATRX and IS10-GFP-ATRX products are highlighted with arrows. Amido black staining of histones was used as a loading control (bottom). A truncated product is observed in the IS10-GFP-ATRX transfections.